Bottom Line:
MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1).We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins.

Affiliation: Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom.

ABSTRACT

Background: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.

Methodology/principal findings: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.

Conclusions/significance: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.

pone-0017270-g007: Binding of different MAGE proteins to NSE4a and NSE4b/EID3 proteins.(A) Cell-free extracts of mouse testes were immunoprecipitated with either IgG or anti-NSE4b followed by Western blotting with the indicated antibodies. (B) S-tagged MAGEA1 or MAGEG1 were co-expressed with FLAG-tagged NSE4b in HEK293 cells. In lanes 1–6 and 13–18, extracts were precipitated with S-protein, whereas in lanes 7–12 they were immunoprecipitated with anti-FLAG antibody. (C–H) S-tagged Class I MAGE protein A1 (C), and class II MAGE proteins D4b (D), F1 (E), G1 (F), necdin (G) or vector alone (H) were co-transfected with FLAG-tagged NSE4a (N4a) (lanes 1–3) or NSE4b/EID3 (N4b) (lanes 4–6). Extracts were immunoprecipitated with S-protein and Western blotted with S-HRP and anti-FLAG antibody.

Mentions:
Of the two orthologs of Nse4, only NSE4a was detected in the SMC5-6 complex from cultured cells, but we showed that, when overexpressed following transfection, either paralog could be incorporated into the complex [18]. Examination of EST libraries suggested that NSE4b was expressed mainly in the testis and tissue-specific micro-array data show that, in the mouse, it is expressed exclusively in testis (http://biogps.gnf.org/#goto=genereport&id=493861). This raised the possibility that it might be the SMC5-6 kleisin in the testis. To test this directly, we used antibodies against NSE4b for immunoprecipitations from extracts of mouse testes. The immunoprecipitates were analysed for other components of the SMC5-6 complex by immunoblotting with anti-hSMC6 and anti-hNSE2/MMS21. Figure 7A, lane 5, shows that both mSMC6 and mNSE2/mMMS21 were co-immunoprecipitated. Finally we immunoprecipitated SMC6 from mouse testes and analysed the immunoprecipitates by mass spectrometry. NSE4b (as well as NSE4a) and other expected members of the complex were detected (data not shown), confirming that NSE4b is a testis-specific component of SMC5-6.

pone-0017270-g007: Binding of different MAGE proteins to NSE4a and NSE4b/EID3 proteins.(A) Cell-free extracts of mouse testes were immunoprecipitated with either IgG or anti-NSE4b followed by Western blotting with the indicated antibodies. (B) S-tagged MAGEA1 or MAGEG1 were co-expressed with FLAG-tagged NSE4b in HEK293 cells. In lanes 1–6 and 13–18, extracts were precipitated with S-protein, whereas in lanes 7–12 they were immunoprecipitated with anti-FLAG antibody. (C–H) S-tagged Class I MAGE protein A1 (C), and class II MAGE proteins D4b (D), F1 (E), G1 (F), necdin (G) or vector alone (H) were co-transfected with FLAG-tagged NSE4a (N4a) (lanes 1–3) or NSE4b/EID3 (N4b) (lanes 4–6). Extracts were immunoprecipitated with S-protein and Western blotted with S-HRP and anti-FLAG antibody.

Mentions:
Of the two orthologs of Nse4, only NSE4a was detected in the SMC5-6 complex from cultured cells, but we showed that, when overexpressed following transfection, either paralog could be incorporated into the complex [18]. Examination of EST libraries suggested that NSE4b was expressed mainly in the testis and tissue-specific micro-array data show that, in the mouse, it is expressed exclusively in testis (http://biogps.gnf.org/#goto=genereport&id=493861). This raised the possibility that it might be the SMC5-6 kleisin in the testis. To test this directly, we used antibodies against NSE4b for immunoprecipitations from extracts of mouse testes. The immunoprecipitates were analysed for other components of the SMC5-6 complex by immunoblotting with anti-hSMC6 and anti-hNSE2/MMS21. Figure 7A, lane 5, shows that both mSMC6 and mNSE2/mMMS21 were co-immunoprecipitated. Finally we immunoprecipitated SMC6 from mouse testes and analysed the immunoprecipitates by mass spectrometry. NSE4b (as well as NSE4a) and other expected members of the complex were detected (data not shown), confirming that NSE4b is a testis-specific component of SMC5-6.

Bottom Line:
MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1).We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins.

Affiliation:
Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom.

ABSTRACT

Background: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.

Methodology/principal findings: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.

Conclusions/significance: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.